Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Dengue virus is the leading arthropod-transmitted viral disease in the world with approximately 390 million human infections per year (1). Nearly 3.6 billion people live in at risk areas for infection, and the global distribution of the two mosquito species that carry the virus (Aedes aegypti and Aedes albopictus) is expanding beyond tropical regions and reaches as far north as New York in North America (2). Primary infection by one of the four Dengue virus serotypes (DENV1-4) typically causes a significant but self-limiting febrile illness, whereas secondary infections can lead to severe disease characterized by hemorrhagic fever and shock syndrome (Severe Dengue or Dengue Hemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS)). These latter syndromes occur in a minor fraction (1% or less) of secondary infections but lead to hospitalization and, in some cases, death. DHF and DSS are thought to arise from a process known as antibody-dependent enhancement (ADE) of infection. In an increasingly accepted model, ADE is caused by antibodies elicited during the course of primary infection that may be potently neutralizing against the primary infection serotype, but also have some cross-reactivity or weak neutralization potential against other serotypes (3). During secondary infection by a heterologous DENV serotype, these antibodies promote uptake and infection of the un-neutralized virus in Fc-γ receptor (FcγR) expressing cells, ultimately increasing viremia. This leads to greater levels of pro-inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6, IFN-γ) and the viral NS1 protein in serum, both of which compromise junctional integrity of capillary endothelial cells (3). Structural proteins encoded by the DENV genome diverge by as much as 40% in amino acid sequence among the four serotypes, and within each of the serotypes, individual genotypes vary by ˜3%. Thus a critical objective for Dengue virus vaccine design is to elicit a broadly neutralizing antibody response against all four serotypes, since weakly cross-reactive antibodies may actually increase the risk of ADE.
Three Dengue vaccine candidates are in clinical development, all of which consist of tetravalent mixtures of attenuated or chimeric viruses. In recently published phase III trials, Sanofi's Dengvaxia®, a tetravalent mixture of yellow-fever virus vector containing DENV1-4 glycoprotein, provided only partial efficacy (<70%) in seropositive cases, and was not effective at all for naïve individuals (4). Nonetheless, Dengvaxia® was recently approved for use in Mexico, the Philippines, Brazil and several other countries in children over the age of 9 who are presumably already flavivirus immune. Two other candidate vaccines are in moving into phase III trials (DENVax, Takeda; and TV003/TV005, NIAID); yet, both also elicited incomplete levels of neutralizing antibody responses (5, 6). Therefore, there is significant rationale for development of alternative vaccine platforms for use either as next-generation primary vaccines, or as boosting agents to improve the efficacy of existing live virus vaccines.
The mature, prefusion glycoprotein E exists as a head-to-tail dimer organized into rafts with icosahedral geometry on the viral particle (7, 8). Each E subunit contains three domains, DI, DII, and DIII. DII contains the fusion loop that inserts into the host cell upon initiation of the fusion reaction in the endosome; DI acts as a rigid connector to DIII, which is anchored via the stem and C-terminal TM domain into the viral membrane. The post-fusion E structure is a trimer with the DIII domain and stem region significantly relocated relative to DI and DII, so as to bring the host and viral membranes into proximity to facilitate viral membrane fusion (9). A host receptor has yet to be identified, but there is circumstantial evidence that interactions between cellular components and DIII initiate attachment and infection (10-12). Neutralizing antibodies arising during infection target a variety of epitopes on the E glycoprotein. Potent and cross-neutralizing antibodies appear to be directed toward either complex quaternary epitopes whose constituents involve portions of the E domains on adjacent dimer subunits (13, 14), or toward the lateral ridge on DIII formed by the A and G strands (15, 16). One example of a DIII-specific broadly neutralizing antibody (bNAb) is the murine mAb 4E11 that potently neutralizes DENV1-3 and weakly neutralizes DENV4 (see ref. (15) for the crystal structure of the DIII-4E11 complex). Recently, high-throughput mutagenesis (“combinatorial alanine scanning”) was used to quantify energetic contributions of contact residues on DIII from all four serotypes recognition feature for 4E11 (17).
Immunization of mice and non-human primates with recombinant DIII constructs (EDIIIs) leads to strong antibody responses, but these antibodies are poorly neutralizing or limited in breadth (18-28). In mice, the immunodominant regions of DIII appear to be in the AB- and FG-loops; resulting monoclonal antibodies are either cross-reactive and non-neutralizing (AB-loop) or type-specific and variably neutralizing (FG-loop) (26, 27). Antibodies that target other domains or more complex epitopes predominate in the human response during the course of natural infection (13, 14, 29, 30). Immunization of non-human primates with EDIII generates a high DIII-specific antibody titer (19, 23, 28). Other immunogen strategies that focus on more complex epitopes or on mimicking the prefusion E dimer are being explored (31), but EDIII has the advantage of being relatively small and easy to produce in large quantities. Dengue EDIII has high potential as an immunogen target, but previous attempts to improve its qualities have not been successful. One strategy to decrease the complexity of tetravalent cocktails is to produce EDIII fusion proteins linking EDIIIs from the four serotypes by flexible linkers (“beads on a string”), but this approach resulted in an imbalanced neutralizing titer response in mice and only partial protection in a suckling mice model for DENV1, 2, and 4 (25). Another strategy is engineering of a “consensus” DIII, in which conserved segments were emphasized (23). However, this approach led to DENV2-specific responses in non-human primates.
The present invention addresses the need for improved methods for preventing and treating Dengue virus infections by providing protein immunogens based on the Dengue virus glycoprotein subunit E domain III (EDIII).